PROJECT ABSTRACT Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in the world, and is an increasing cause of significant morbidity and mortality. Despite its growing impact on global health there are currently no FDA-approved therapies for treatment. While the pathophysiology of NAFLD is complex, increasing evidence supports a direct role of alterations in hepatic NADH/NAD+ levels. However, testing the role of NADH/NAD+ in hepatic physiology is challenging given the limitations of existing tools with which to manipulate it in a precise manner, and the lack of circulating biomarkers for NADH/NAD+ which, as unstable intracellular metabolites, would be impractical to measure in most clinical and human research settings. The Mootha lab has developed a genetically encoded metabolic tool, called LbNOX, to directly and precisely manipulate cellular NADH/NAD+ in different tissues and cellular compartments. In preliminary work, I have used this tool both in vitro and in vivo to identify that a circulating metabolite, ?-hydroxybutyrate (?HB) is sensitive to alterations in hepatic NADH/NAD+. ?HB, has previously been shown to be a biomarker of insulin resistance and sensitive to polymorphisms in the gene GCKR, which in turn has been associated with NAFLD. Our preliminary work also shows LbNOX improves hepatic insulin resistance in vivo, and that GCKR influences hepatocyte NADH/NAD+. These findings link GCKR, NAFLD, ?HB, and hepatic NADH/NAD+. The central hypothesis of this proposal is that ?HB is a circulating biomarker of hepatic NADH/NAD+ which is causally linked to hepatic insulin resistance and steatosis, and that genetic modulators of hepatic NADH/NAD+ influence hepatic steatosis and insulin resistance In this proposal, I will use a combination of in vitro and in vivo hepatic LbNOX expression to further define the causal connection between hepatic insulin resistance, hepatic steatosis, and hepatic NADH/NAD+. I will define the mechanism by which LbNOX improves hepatic insulin resistance, and the mechanism by which GCKR influences hepatic NADH/NAD+. I am a clinical and research hepatologist dedicated to a research career as a physician scientist specializing in metabolic aspects of liver disease. The proposed research plan will allow me to develop new knowledge and expertise in metabolism, metabolomics, and hepatic physiology, and provide experience with animal models of chronic liver disease. Throughout the proposed research I will be guided by a formal research advisory committee comprised of outstanding mentors and experts in metabolism, NAFLD, and hepatic physiology, all in the setting of a stellar research environment comprised of MGH, the Broad Institute, and affiliated institutions. The research proposed, along with the guidance of my mentors and collaborators in this research, will help ensure my successful transition to scientific independence.